Satoh Keitaro, Matsuki-Fukushima Miwako, Qi Bing, Guo Ming-Yu, Narita Takanori, Fujita-Yoshigaki Junko, Sugiya Hiroshi
Dept. of Physiology, Nihon Univ. School of Dentistry at Matsudo, 2-870-1 Sakaecho-nishi, Matsudo, Chiba 271-8587, Japan.
Am J Physiol Gastrointest Liver Physiol. 2009 Jun;296(6):G1382-90. doi: 10.1152/ajpgi.90536.2008. Epub 2009 Apr 16.
Myristoylated alanine-rich C kinase substrate (MARCKS) is known as a major cellular substrate for protein kinase C (PKC). MARCKS has been implicated in the regulation of brain development and postnatal survival, cellular migration and adhesion, as well as phagocytosis, endocytosis, and exocytosis. The involvement of MARCKS phosphorylation in secretory function has been reported in Ca(2+)-mediated exocytosis. In rat parotid acinar cells, the activation of beta-adrenergic receptors provokes exocytotic amylase release via accumulation of intracellular cAMP levels. Here, we studied the involvement of MARCKS phosphorylation in the cAMP-dependent amylase release in rat parotid acinar cells. MARCKS protein was detected in rat parotid acinar cells by Western blotting. The beta-adrenergic agonist isoproterenol (IPR) induced MARCKS phosphorylation in a time-dependent manner. Translocation of a part of phosphorylated MARCKS from the membrane to the cytosol and enhancement of MARCKS phosphorylation at the apical membrane site induced by IPR were observed by immunohistochemistry. H89, a cAMP-dependent protein kinase (PKA) inhibitor, inhibited the IPR-induced MARCKS phosphorylation. The PKCdelta inhibitor rottlerin inhibited the IPR-induced MARCKS phosphorylation and amylase release. IPR activated PKCdelta, and the effects of IPR were inhibited by the PKA inhibitors. A MARCKS-related peptide partially inhibited the IPR-induced amylase release. These findings suggest that MARCKS phosphorylation via the activation of PKCdelta, which is downstream of PKA activation, is involved in the cAMP-dependent amylase release in parotid acinar cells.
肉豆蔻酰化富含丙氨酸的蛋白激酶C底物（MARCKS）是蛋白激酶C（PKC）的主要细胞底物。MARCKS与脑发育和出生后存活、细胞迁移和黏附以及吞噬作用、胞吞作用和胞吐作用的调节有关。在Ca(2+)介导的胞吐作用中，已报道MARCKS磷酸化参与分泌功能。在大鼠腮腺腺泡细胞中，β-肾上腺素能受体的激活通过细胞内cAMP水平的积累引发淀粉酶的胞吐释放。在此，我们研究了MARCKS磷酸化在大鼠腮腺腺泡细胞中cAMP依赖性淀粉酶释放中的作用。通过蛋白质印迹法在大鼠腮腺腺泡细胞中检测到MARCKS蛋白。β-肾上腺素能激动剂异丙肾上腺素（IPR）以时间依赖性方式诱导MARCKS磷酸化。通过免疫组织化学观察到，一部分磷酸化的MARCKS从膜易位到细胞质中，并且IPR诱导顶端膜位点的MARCKS磷酸化增强。cAMP依赖性蛋白激酶（PKA）抑制剂H89抑制了IPR诱导的MARCKS磷酸化。PKCδ抑制剂rottlerin抑制了IPR诱导的MARCKS磷酸化和淀粉酶释放。IPR激活了PKCδ，并且IPR的作用被PKA抑制剂抑制。一种与MARCKS相关的肽部分抑制了IPR诱导的淀粉酶释放。这些发现表明，通过激活PKCδ（其在PKA激活的下游）的MARCKS磷酸化参与了腮腺腺泡细胞中cAMP依赖性淀粉酶的释放。
